Administration of donor antigen to adult hosts conditioned by immunosuppressive therapy, represents one strategy to produce central immunologic tolerance, leading to host acceptance of foreign transplants. In contrast to their cell-borne counterparts, extracted histocompatibility antigens (e-HAg) proffer not only non-replicating materials bearing a relatively limited array of disparate determinants, but also a soluble state that may display uniquely exposed native, or chemically manipulated artificial, epitopes facilitating tolerance induction. Initial studies revealed the combination of peri-operative therapy with 3M KC1 BUF e-HAg and CsA (10mg/kg/day) to prolong the survival of WFu rat renal, cardiac and pancreatic islet grafts (major plus minor histocompatibility differences), as well as mongrel canine kidney transplants. Alternatively, pre-transplant conditioning with 1600 R total lymphoid irradiation (TLI) followed by e-HAg prolonged rat heart graft survival. On the one hand, the proposed studies seek to improve the e-HAg stimulus by purification of active epitopes, including separation of Class I versus Class II major histocompatibility complex determinants, and by optimization of the route, dose, form and infusion technique. On the other hand, these studies dissect the immunologic effects of e-HAg therapy combined with either CsA or TLI, using limiting dilution analysis to assess precursor frequency of helper, cytotoxic and suppressor T-lymphocyte subpopulations at serial times starting pre-and continuing to 180 days post-transplant. To reveal the target of their action, e-HAg induced suppressor cells are panned on cellular monolayers of or stimulated in proliferation assays with antigen-or idiotype-specific markers. Based upon initial studies showing e-HAg triggers specific suppressor cells, these elements will be cloned, in order to dissect their mechanism of action, specificity, and in vivo and in vitro potency. Finally, the proposed experiments analyze the effects of new immunosuppressive modalities, namely monoclonal rat anti-delta-interferon antibody and continuous intra-arterial CsA infusion, both of which were discovered during the initial grant period, on allograft H-antigen expression, suppressor cell induction, and efferent protection against attack by effector lymphocytes. These studies may provide a foundation for application of e-HAg with selective immunosuppressive adjuncts in preclinical tests, and eventually for clinical organ transplantation.